Choline Esters

ABSTRACT

Compounds, formulations, and methods are provided containing the choline ester of a reducing agent, especially lipoic acid or derivatives thereof. The compounds may be administered via a topical ocular route to treat or prevent oxidative damage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.61/187,005 filed Jun. 15, 2009, 61/224,930 filed Jul. 13, 2009, and61/242,232 filed Sep. 14, 2009, each of which is incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

As we age, our lenses undergo physiological changes that make it moredifficult to focus on near objects. That is why nearly everyone requiresreading glasses, even as early as age 35-40. The ability of the eye tochange focal power, also known as accommodative amplitude, decreasessignificantly with age. The accommodative amplitude is 20 diopters inchildren and young adults, but it decreases to 10 diopters by age 25 andto ≤1 diopter by age 60. The age-related inability to focus on nearobjects is called presbyopia. All of us will develop presbyopia and willuse corrective lenses unless a new treatment is found.

Both presbyopia and cataract are age-related and may share commonetiologies such as lens growth, oxidative stress, and/or disulfide bondformation.

There is a need for compositions, formulations and methods for combatingpresbyopia and/or cataract, particularly compositions and methods thatminimize toxicity to surrounding healthy tissues.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, a compound is provided that is the choline ester oflipoic acid or a derivative of lipoic acid. In one embodiment, thelipoic acid is alpha lipoic acid. In another embodiment, the derivativeof lipoic acid is: 6,8-dimercaptoctanoic acid; dihydrolipoate;5-(1,2-thiaselenolan-5-yl) pentanoic acid; or 5-(1,2-thiaselenolan-3-yl)pentanoic acid. The lipoic acid or derivative of lipoic acid can includethe R enantiomer.

In another embodiment, a pharmaceutical composition is providedcomprising an active agent that is a reducing agent-choline ester atleast one pharmaceutically acceptable excipient. In one embodiment, thereducing agent lipoic acid or a derivative thereof, e.g., lipoic acidcholine ester. The active agent can be present in an amount of about0.1% to about 10% more specifically about 0.5% to about 10%.

In one embodiment, the pharmaceutical composition includes a buffer, atonicity agent, and/or a viscosity agent. In one embodiment, the bufferis a phosphate buffer. In another embodiment, the viscosity agent is acellulosic agent.

In one embodiment, the pharmaceutical composition includes a biochemicalenergy source, e.g., pyruvate or alanine.

In one embodiment, the pharmaceutical composition has a pH of about 4 toabout 7.5. In another embodiment, the pharmaceutical composition has apH of about 5 to about 6.

In one embodiment, the pharmaceutical composition is suitable fortopical ocular delivery, e.g., an eye drop.

In one embodiment, a pharmaceutical composition is provided thatcontains:

-   -   about 0.25% to about 10% of a reducing agent-choline ester,    -   optionally, about 0.05% to about 1.0% of a biochemical energy        source,    -   about 0.25% to about 1% buffer,    -   about 0.2% to about 0.6% tonicity agent, and    -   about 0.1% to about 0.4% viscosity agent.

In another embodiment, a pharmaceutical composition is provided thatcontains:

-   -   5% lipoie acid choline ester,    -   0.1% ethyl pyruvate,    -   0.269% sodium phosphate monobasic inoriohydrate,    -   0.433 sodium phosphate dibasic anhydrous,    -   0.5% sodium chloride, and    -   0.2% hydroxypropyl rnethylcellulose.

In another embodiment, a pharmaceutical composition is provided thatcontains:

-   -   5.0% lipoic acid choline ester,    -   0.5% alanine,    -   0.269% sodium phosphate monobasic monohydrate,    -   0.433% sodium phosphate dibasic anhydrous,    -   0.5% sodium chloride, and    -   0.2% hydroxyprivyl methyl cellulose.

In yet another embodiment, a method of preventing or treating oxidationdamage to cells is provided by administering the pharmaceuticalcomposition. The method can optionally include administering abiochemical energy source.

In one embodiment, the cells are in vivo. In another embodiment, thecells are ocular cells.

In one embodiment, administering is administering by topical oculardelivery.

In another embodiment, a method is provided for a one-step synthesiscomprising reacting a reducing agent (e.g., lipoic acid) with ahalogenated choline (e.g., bromocholine bromide) to yield a cholineester.

In another embodiment, a small portion of the DHLA-thiolactone can reactwith low pK lysine protein residues to form a post-translationalacylation product, denoted as Nepsilon-lipoyl group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the accommodative amplitude in diopters (D) of anuntreated human lens as a function of age in years. Bonja, D et at.2008. Optical Power of the Isolated Human Crystalline Lens. InvestOphthalmol Sci 49(6):254 1-8. Borja et al. calculated the maximumpossible accommodative amplitude of each measured lens power data point(n=65). As shown, there is good agreement between the age-dependent lossof accommodation and the maximum amplitude of accommodation calculatedfrom the isolated lens power.

FIG. 2 shows a trend graph of the shear modulus versus position in thelens and age. Weeber, et at. 2007. Stiffness gradient in the crystallinelens. Graefes Ache Clin Exp Ophthalmol 245(9):1357-66. The line at thebottom is the 20-year-old lens; the line at the top is the 70-year oldlens. The modulus increases with age for all positions in the lens.Measurements were taken up to 4.0 mm from the lens centre. The lines areextrapolated to a radius of 4.5 mm (lens diameter 9.0 mm).

FIG. 3 depicts the average opacity (opacimetry) of an untreated humanlens as a function of age in years. Bonomi, L′ t al. 1990. Evaluation ofthe 701 interzeag lens opacity meter. Grafe's Arch Clin Exp Ophthalmol228(5):447-9. Lens opacity was measured in 73 healthy subjects between10 and 76 years of age without slit-lamp evidence of cataract and with avisual acuity of 20/20. These subjects were classified into ten agegroups. This study was carried out using the Interzeag Opacity Meteraccording to the procedure described by Flammer and Bebies (Flammer J,Bebie H. 1987, Lens Opacity Meter: a new instrument to quantify lensopacity. Ophthalmological 195(2):69-72) and following the suggestions ofthe operating manual for the instrument.

FIG. 4 depicts a scatter plot of the change in ΔD (micrometers) in theabsence (control) and presence of lipoic acid in lens organ cultureexperiments. The symbol ‡ designates significantly larger changes in ΔDwhen compared to controls. Statistical values are highly significant atp<0.0001 by unpaired t-test and by Kruskal Wallis test, which comparedmedians of each data set. The relative change is Young's modulus (E) canbe calculated as the cubic value derived from the ΔD of the controldivided by the ΔD of the experimental or E fractional change=(ΔDcon/ΔDexp)̂3.

FIG. 5 depicts a scattergram of the percent of the total protein SHgroups in disulfide bonds. Free SH groups were alkylated with4-acetamido-4′-maleimidylstilbene-2,2′-sulfonic acid (c, 1 μM, 5 μM, 9.6μM, 50 μM, 96 μM) or 7-diethylamino-3-(4′ maleimidylphenyl)-4-methylcoumarin (500 μM, and 500 μM c). Following removal of the firstalkylating agent, the S-S bonds were reduced and alkylated withfluorescein-5-maleimide. Absorption spectra were used to calculatedtotal protein (A280 nm), free protein SH (A322 or A384), and protein SS(A490) using the appropriate extinction coefficients. The symbol ‡indicates statistically significant difference of mean with mean ofcontrol (c, p≤0.05). The symbol ** indicates means of 500 μM lipoic acidand the 500 μM control were significantly different from each other(p=0.027).

DETAILED DESCRIPTION OF THE INVENTION

Compounds, formulations, and methods are provided that can prevent,reduce, reverse, and/or slow the rate of lens growth, oxidative damage,and/or disulfide bond formation. These compounds, formulations, andmethods may thus effectively prevent or treat presbyopia and/orcataract.

The compounds, formulations, and methods described herein employ anactive agent that is the choline ester of a reducing agent.

Reducing Agents

The reducing agent is capable of reducing disulfide bonds, particularlydisulfide bond formation in lens membranes and membrane associatedproteins. Accordingly, particularly preferred reducing agents arecapable of entering into the lens epithelial cells.

In one embodiment, the reducing agent enters the lens epithelial cellsusing a naturally occurring transport mechanism. For example, lipoicacid enters lens cells via specific plasma membrane symporters andantiporters. In one embodiment, the reducing agent is a derivative oflipoic acid that while not structurally identical to acid, neverthelessmaintains the capability of utilizing the naturally occurring transportmechanism the lipoic acid.

In one embodiment, the reducing agent is lipoic acid or a derivativethereof. In some embodiments, the reducing agent is alpha lipoic acid ora derivative thereof. In one embodiment, the reducing agent is lipoicacid per se (5-(1,2-dithiolan-3-yl)pentanoic acid), e.g., alpha lipoicacid.

In another embodiment, the reducing agent is a lipoic acid derivative.Lipoic acid derivatives include, but are not limited to,6,8-dimercaptoctanoic acid (dihydrolipoic acid) and dihydrolipoate.Lipoic acid derivatives also include seleno-substituted lipoic acidderivatives including, but not limited to,5-(1,2-thiaselenolan-5-yl)pentanoic acid and5-(1,2-thiaselenolan-3-yl)pentanoic acid.

In another embodiment, the reducing agent can be any of the reducingagents described in co-pending U.S. patent application Ser. Nos.11/946,659, 12/267,260, or 12/390,928.

Choline Esters

The reducing agent as described above may be provided as a cholineester. Without being bound by theory, it is believed that the cholineester may improve the agent's solubility in pharmaceutical formulations.It may also improve corneal permeability.

In one embodiment, the active agent is the choline ester of lipoic acid,e.g., alpha lipoic acid, or a lipoic acid derivative. In one embodiment,the active agent is lipoic acid choline ester. In another embodiment,the active agent is alpha lipoic acid choline ester.

The structure may include a counterion, wherein the counterion is anypharmaceutically acceptable counterion capable of forming a salt. In yetanother embodiment, the active agent is the choline ester of a lipoicacid derivative.

Any of the reducing agents can be prepared as a pharmaceuticallyacceptable salt. The term “pharmaceutically acceptable salt” includessalts of the active compounds that are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorus acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, furnaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolysulfonic, citric, tartaric, oxalic, methanesulfonic, and the like.Also included are salts of amino acids such as arginate and the like,and salts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977 , 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Examples of such salts include, but are limited to,hydrochlorides, hydrobromides, sulfates, methancsulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, mixtures thereof including racemic mixtures), succinates,benzoates, and salts with amino acids such as glutamic acid. These saltsmay be prepared by methods known to those skilled in the art.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In one embodiment, the counterion ion is the1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium cation (i.e.,tromethamine salt).

Pharmaceutical Formulations

The active agent can be combined with one or more pharmaceuticallyacceptable excipients to form a pharmaceutical composition. In thepharmaceutical compositions herein, the active agent may be present asthe choline ester.

The active agent can be administered as a racemate or as an coantiomer.Lipoic acid and its derivatives are preferably administered to includethe R form. Synthetic methods to yield a racemate may be less expensivethan stereo-specific processes including isolation/purification steps.On the other hand, administering a single enantiomer can lower thetherapeutically effective amount, thus decreasing any toxicity effectsof the active agent.

As the agents described herein may have therapeutic uses as described infurther detail below, it is preferable to select an active agent withlow toxicity. Additional acceptable lipoic acid derivatives can beselected by in vitro toxicology testing.

The amount of the active agent (e.g., the reducing agent-choline ester)in the pharmaceutical formulation can be selected based on the conditionof the subject to be treated, including the subject's age, gender, aswell as vision and lens status. Exemplary amounts of the active agentcan be about 0.25% to about 10%, about 0.5% to about 10%, about 1% toabout 8%, about 3% to about 7%, about 2% to about 5%, about 5% to about7%, or about 5%. In another embodiment, the amount of active agent isless than about 0.1% (100 mg) or up to about 10% (10000 mg).

In one embodiment, the pharmaceutical composition is formulated forocular use. Ocular formulations include, but are not limited to, liquidformulations (e.g., solutions, suspensions) for topical administrationas well as formulation for injection or ocular insert administration.Preferably, the ocular formulation is formulated for topicaladrninistration such as an eye, drop, swab, ointment, gel, or mist (e.g,an aerosol or spray). In one embodiment, the formulation is an eve drop.For ocular formulations, the pharmaceutically acceptable exciptonts areselected to be corripatible with, and suitable for, ocular use. Suchexcipients are well known in the art. In one embodiment, excipients maybe selected to improve the solubility of the agent.

Exemplary excipients include, but are not limited to, buffers, tonicityagents, viscosity agents, preservatives, emulsifiers, salts, lubricants,polymers, solvents, and other known excipients for ocular pharmaceuticalformulations. Appropriate amounts can be determined by one of ordinaryskill in the art, but non-limiting exemplary amounts (in % by weight)are also provided below.

In one embodiment, the pharmaceutical composition includes one or morebuffers to adjust or maintain the pH of the formulation. In oneembodiment, the pH is near physiological pH (pH of tears is about 7).Thus, the pH of the formulation can be about 6 to about 8, about 6.5 toabout 7.5, about 6.8 to about 7.2, about 7.1 to about 7.5, or about 7.In another embodiment, the pH is about 5.5. Thus, the pH of theformulation can be about 4 to about 7, about 4.5 to about 6, about 4.5to about 5.5, about 5.5 to about 6.5, about 5 to about 6, about 5.25 toabout 5.75, or about 5.5. Exemplary buffers include, but are not limitedto, phosphate buffers (e.g., sodium phosphate monobasic monohydrate,sodium phosphate dibasic anhydrous), borate buffers, and HBSS (Hank'sBalanced Salt Solution). In one embodiment, the buffer is a phosphatebuffer. In another embodiment, the buffer is sodium phosphate monobasicmonohydrate and/or sodium phosphate dibasic anhydrous. The buffer amount(amount of either total buffer or a single buffer excipient) can be 0.1%to about 1.0%, about 0.2% to about 0.6%, about 0.05% to about 0.5%,about 0.25% to about 0.45%, or about 0.25%, about 0.43%, or about 0.7%.In one embodiment, the buffer is about 0.05% to about 0.5% (e.g., about0.27%) sodium phosphate monobasic monohydrate and about 0.2% to about0.6% (e.g., about 0.43%) sodium phosphate dibasic anhydrous.

In one embodiment, the pharmaceutical composition includes one or moretonicity agents. Although the formulation may be hypertonic orhypotonic, isotonic formulations are preferred (260-320 mOsm). Exemplarytonicity agents include, but are not limited to, sodium chloride. Thetonicity agent amount can be about 0.1% to about 5%, about 0.1% to about2%, about 0.1% to about 1% about 0.25% to about 0.75%, about 0.2% toabout 0.6%, or about 0.5%, In one embodiment, the tonicity agent isabout 0.2% to about 0.6% (e.g., about 0.5%) sodium chloride.

In one embodiment the pharmaceutical composition includes one or moreviscosity agents to increase the viscosity of the formulation. Exemplaryviscosity agents include, but are not limited to, cellulosic agents(e.g., hydroxypropyl methylcellulose), polycarbophil, polyvinyl alcohol.In one embodiment, the viscosity agent is a cellulosic agent, e.g.,hydroxypropyl methylcellulose. The visocosity agent amount can be about0.1% to about 5%, about 0.1% to about 2%, about 0.1% to about 1%, about0.1% to about 0.4%, or about 0.2%. In one embodiment, the viscosityagent is about 0.1% to about 0.4% (e.g., about 0.2%) hydroxypropylmethylcellulose.

In one embodiment, the pharmaceutical composition includes one or morepreservatives to minimize microbial contamination or enhance shelf life.Exemplary preservatives include, but are not limited to, benzalkoniumchloride (BAK), cetrimonium, chlorobutanol, edetate disodium (EDTA),polyquaternium-1 (Poluquad®), polyhexamethylene biguanide (PHMB),stabilized oxychloro complex (PURITE®), sodium perborate, and SofZia®.The preservative amount amy be, e.g., less than about 0.02%, about0.004% or less, or about 0.005% to about 0.01%.

In one embodiment, the pharmaceutical composition includes one or morestabilizers. Exemplary stabilizers include, but are not limited to aminoacids such as alanine. The stabilizer amount can be about 0.1% to about5%, about 0.1% to about 2%, about 0.1% to about 1%, about 0.25% to about0.75%, about 0.2% to about 0.6%, or about 0.5%. In one embodiment, thestabilizer is about 0.2% to about 0.6% (e.g., about 0.5%) alanine.

In one embodiment, the pharmacceutical composition includes one or moreemulsifiers. Exemplary emulsifiers include, but are not limited to,Polysorbate 80.

The compounds described herein can be used in combination with oneanother, with other active agents known to be useful in ocular disese,or with adjunctive agents that may not be effective alone, but maycontribute to the efficacy of the active agent. For example, adjunctiveagents might include one or more amino acids or choline (separate fromthe lipoic acid compound) to enhance the efficacy of the active agent.The combinations can be advantageous, e.g., in reducing metabolicdegradation.

The term “co-administer” means to administer more than one active agent,such that the duration of physiological effect of one active agentoverlaps with the physiological effect of a second active agent. In someembodiments, co-administration includes administering one active agentwithin 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of a secondactive agent. Co-administration includes administering two active agentssimultaneously, approximately simultaneously (e.g., within about 1., 5.10, 15, 20, or 30 minutes of each other), or sequentially in any order.In some embodiments, co-administration can be accomplished byco-formulation, i.e., preparing a single pharmaceutical compositionincluding both active agents. In other embodiments, the active agentscan be formulated separately. In another embodiment, the active and/oradjunctive agents may be linked or conjugated to one another.

Without being bound by theory, it is believed that the administration ofan active agent, e.g., lipoic acid or a derivative thereof, and anadjunctive agent such as choline, can be particularly advantageous in aconjugated form. The conjugate compound be applied to the cornea, andpenetration is achieved due to the bi-phasic (water and lipid soluble)nature of the conjugate compound. As the conjugate goes through thecornea, naturally present esterases (enzymes) separate lipoic acid fromcholine. The lipoic acid (now a pro-drug) in the aqueous bathes the lensand enters the lens epithelial cells (due to low molecular weight andsize), and there is reduced by any one of several oxido-reductases(enzymes such as thioredoxin and thioltransferase) to form dihydrolipoicacid. Dihydrolipoic acid now has two extra hydrogen atoms to donate to adisulfide complex (e.g., protein disulfide PSSP), separating the twosulfur atoms into sulfhydril molecules (e.g., protein cysteine residuesPSH with free SH groups) thus breaking the inter-cytosol proteincross-links. Breaking these cross-link is what reduces the lensstiffness. Once donation of the hydrogen atoms to the sulfur atom, thedihydrolipoic acid becomes lipoic acid and is available for recycling inthe cell to become dihydrolipoic acid or converted to a natural degradedby product thiolactone and excreted.

In one embodiment, a reducing agent, such as one of the compoundsdescribed herein, is co-administered with a biochemical energy source. Abiochemical energy source facilitates reduction by participating as anintermediate of energy metabolic pathways, particularly the glucosemetabolic pathway. Exemplary intermediates of this pathway are depictedby, e.g., Zwingmann, C. et al. 2001, 13C Isotopomer Analysis of Glucoseand Alanine Metabolism Reveals Cytosolic Pyruvate Compartmentation asPart of Energy Metabolism in Astrocytes, GLIA 34:200-212. Exemplarybiochemical energy sources include, e.g., glucose or a portion thereof(e.g., glucose-6-phosphate:(G6P)), pyruvate (e.g., ethyl pyruvate),NADPH, lactate or derivative thereof. G6P may be favored over glucosesince a formulation including glucose may further benefit from theaddition of preservatives. In one embodiment, the biochemical energysource is an intermediate a cytosolic metabolic pathway. Exemplarycytosolic pathway intermediates include, e.g., glucose, pyruvate,lactate, alanine, glutamate, and 2-oxoglutarate. In another embodiment,the biochemical energy source is an intermediate in a mitochondrialmetabolic pathway. Exemplary mitochondrial pathway intermediatesinclude, e.g., pyruvate, TCA-cycle intermediates, 2-oxoglutarate,glutamate, and glutamine. In one embodiment, the biochemical energysource is pyruvate compound (e.g., ethyl pyruvate). In anotherembodiment, the biochemical energy source is alanine. The amount of abiochemical energy source can be, e.g., about 0.05% to about 1.0%. Inone embodiment, the energy source is 0.1% ethyl pyruvate.

In one embodiment, the agent is co-administered with glucose-6-phosphate(G6P). NADPH, or glucose. In one embodiment, the agent is activated byan endogenous chemical energy, e.g., endogenous glucose. For example,endogenous glucose can activate lipoic acid or a derivative thereof todihydrolipoic acid (DHLA) or a corresponding derivative thereof.

In one embodiment, the pharmaceutical formulation includes a reducingagent-choline ester as the active agent and one or more pharmaceuticalexcipients selected from the group consisting of buffers, tonicityagents, and viscosity agents.

The pharmaceutical formulation may be packaged for administration by anymeans known in the art including, but not limited to, individual doseunits or multi-dose units, e.g., dropper bottles. Multi-dose units mayinclude, for example, about 1 mL to about 100 mL, about 1 mL to about 50mL, about 1 to about 10 mL, about 2 mL to about 7 mL, or about 5 mL. Anindividual dose may be, e.g., 1-10 drops, 1-5 drops, or 2-3 drops,wherein each drop is about 5 to about 50 μl, about 10 to about 30 μl, orabout 20 μl. Depending on the active agent concentration and thecondition of the patient, doses may be administered, for example, 1-4,preferably 1-2 times per day.

Methods of Synthesis

Although choline esters may be prepared via a multi-step process asdepicted in Example 3, in one embodiment, a one-step method of synthesisfor the choline esters is provided. The method comprises: providing areducing agent as described above, reacting the reducing agent with ahalogenated choline to yield a choline ester of the reducing agent. Inone embodiment, the halogenated choline bromocholine bromide as follows:

In some embodiments, the reaction is conducted in a solvent, such asacetone or dimethyl formamide (DMF).

In one embodiment, the reaction mixture further includes a base.Exemplary bases include, but are not limited to, K₂CO₃, Cs₂CO₃, KF,NaHCO₃, and KH₂PO₄. The base can be present in an amount of about 1 toabout 5 equivalents relative to the reducing agent. In some embodiments,the base amount is about 1 eq.

Methods of Treating Oxidation Damage

The agents described herein can be employed in a method including thestep of providing a reducing agent-choline ester active agent to a cell,either in vitro or in vivo.

The active agents described herein can be employed in a method fortreating or preventing oxidation damage to cells. Such a method includesthe step of administering a pharmaceutical composition comprising areducing agent-choline ester active agent to a cell, either in vitro orin vivo.

As stated above, the agents can be delivered to cells in vitro or invivo. In one embodiment, the cells are in vivo. In either case, thecells can be ocular cells, e.g., lens cells. In one embodiment, theagent is delivered to a lens, either in vitro or in vivo. In oneembodiment, the compounds described herein can be used in a method fortreating ocular disease. Exemplary ocular diseases include, but are notlimited to: presbyopia, cataract, macular degeneration (includingage-related macular degeneration), retinopathies (including diabeticretinopathy), glaucoma, and ocular inflammations. In one embodiment, theocular disease to be treated is cataract. In another embodiment, theocular disease to be treated is treat presbyopia. Because oxidativedamage has been implicated in other disorders including cancer, theagents may prove useful for administration to any type of cellexhibiting or prone to oxidative damage.

The methods preferably utilize a therapeutically effective amount of theactive agent. The term “therapeutically effective amount” means anamount that is capable of preventing, reducing, reversing, and/orslowing the rate of oxidative damage. For ocular applications, atherapeutically effective amount may be determined by measuring clinicaloutcomes including, but not limited to, the elasticity, stiffness,viscosity, density, or opacity of a lens.

Lens elasticity decreases with age, and is a primary diagnostic andcausative factor for presbyopia. Lens elasticity can be measured asaccommodative amplitude in diopters (D). FIG. 1 depicts the averageelasticity in diopters of an untreated human lens as a function of agein years. The lower the value of D, the less elastic the lens. In oneembodiment, the agents described herein (in the active form) candecrease and/or maintain D at a value that is greater than the D valueexhibited by an untreated lens of about the same age. In other words,the agents can keep accommodative amplitude “above the line” (the solidline mean accommodative amplitude) depicted in FIG. 1. In oneembodiment, D is increased and/or maintained at a value about 2, 5, 7,10, 15, 25, 50, 100, 150, or 200 percent above the line. However, asindividual lenses may differ with respect to average values, anotherembodiment provides any increase in elasticity, maintenance ofelasticity, or reduction in the rate of decline of elasticity (i.e.,reduction in the rate of decrease in diopters) for an individual lenscompared to the elasticity of the same lens before treatment. In anotherembodiment, the methods provide an objective increase in elasticity ofat least about 0.1, 0.2, 0.5, 1, 1.2, 1.5, 1.8, 2, 2.5, 3, or 5diopters.

Lens elasticity can also be measured by the unit of elasticity E. Thehigher the value of E, the less elastic the lens. FIG. 2 depicts theaverage elasticity (E) of an untreated human lens as a function of agein years. In one embodiment, the agents described herein (in the activeform) can decrease and/or maintain E at a value that is less than the Evalue exhibited by an untreated lens of about the same age. In otherwords, the agents can keep lens elasticity “below the line” depicted inFIG. 2. In one embodiment, E is decreased and/or maintained at a valueabout 2, 5, 7, 10, 15, 25, 50, 100, 150, or 200 percent below the line.However, as individual lenses may differ with respect to average values,another embodiment provides any increase inelasticity, maintenance ofelasticity, or reduction in the rate of decline or elasticity (i.e.,reduction in the rate of increase in E value) for an individual lenscompared to the elasticity of the same lens before treatment.

Therapeutic efficacy can also be measured in terms of lens opacity. Lensopacity increases with age and is a primacy diagnostic and causativefactor for cataract. FIG. 3 depicts the average opacity of an untreatedhuman lens as a function of age in years. In one embodiment, the agentsdescribed herein (in the active form) can decrease and/or maintainopacity at a value that is less than the opacity value exhibited by anuntreated lens of about the same age. In other words, the agents cankeep lens opacity “below the line” depicted in FIG. 3. In oneembodiment, lens elasticity is decreased and/or maintained at a valueabout 2, 5, 7, 10, 15, 25, 50, 100, 150, or 200 percent below the line.However, as individual lenses may differ with respect to average values,another embodiment provides any decrease, maintenance, or reduction inthe rate of increase of opacity for an individual lens compared to theopacity of the same lens before treatment.

Some agents described herein exist naturally in the untreated eye.Lipoic acid, for example, occurs naturally in eye tissue. In general, atherapeutically effective amount of the exogenously administered agentis often at least about 1 or 2 orders of magnitude larger than thenatural level of the compound. In one embodiment, the bioavailable tothe lens dose amount of lipoic acid or a derivative thereof is about 5μM to about 250 μM or about 10 μM to about 700 μM. The dose amount willdepend on the route of administration as well as the age and conditionof the patient. Similarly, the frequency of dosing will depend onsimilar factors as can be determined by one of ordinary skill in theart.

Efficacy has been demonstrated in vitro for specific exemplary dosing.(See Example 2). FIG. 2 shows that the inelasticity increases by afactor of nearly 20 during the critical period from age 40 to 55 years.From current data, a 10 μM dose can decrease the inelasticity over 95%within a millimeter volume element (voxel). Extrapolation of theseresults to a volume element in the human lens suggests that using thistreatment dose on a 55 year old person with a 10 kPA lens startingmodulus value (see FIG. 2) could be reduced after treatment to a valueof about 0.5 kPA (which then corresponds to a value typically seen witha 40 yr old person). FIG. 1 permits a conversion of these modulus valuesto optical amplitude: accommodative amplitude is normally reduced toalmost 0 above 55 years, while a person at 40-45 years still exhibitsaround 4-5 diopters of accommodation.

This method includes the description of a topical ocular formulationthat will be used to administer one to two drops of the active agent(s)to the cornea. The formulation will be devised such to providesufficient active agent and effect treatment to the lens. The mechanismof treatment employs using the intrinsic cellular energy to reduce theactive agent lipoate-[S-S] (actually a pro drug) to dihydrolipoate[DHLA-(SH)₂] (the reduced active agent). DHLA is then used to reduceprotein disulfide bonds and alter the lens material properties of thelens to restore accommodative amplitude. The activation of the activeagent lipoate to DHLA is enzymatically formed with endogenousintracellular oxido-reductase, including such enzymes as thioredoxin,lipoamaide dehydrogenase, and glutathione reductase. These enzymes useendogenous NADPH to affect the redox couple and recycle lipoate to thereduced form: DHLA. DHLA to can however undergo additional metabolismwithin the lens to produce a number of other products, including7-(2-mercaptoethyl)thiepan-2-one (henceforth referred to as“DHLA-thiolactone”). A small portion of the DHLA-thiolactone can reactwith low pk lysine protein residues to form a post-translationalacylation product, denoted as Nepsilon-lipoyl group. This laterpost-translation product is normally localized in the mitochondrialsystem and is important with the pyruvate dehydrogenase-acetyltansferaseactivity. Any excess DHLA-thiolactone is released into the aqueous alongwith DHLA itself and other byproducts. At 15 minutes to 2 hours aftertopical dosing, the amount of DHLA-thiolactone measured in the aqueousranges from 10 micro molar levels to 700 micro molar levels.

The methods include preventative methods that can be performed onpatients of any age. The methods also include therapeutic methods thatcan be performed on patients of any age, particularly patients that are20, 25, 30, 35, 40, 45, 50, 52, 55, 57, 60, 70, 75, or 80 years of ageor older.

Any numerical values recited herein include all values from the lowervalue to the upper value, in increments of any measurable degree ofprecision. For example, if the value of a variable such as age, amount,time, percent increase/decrease and the like is 1 to 90, specificallyfrom 20 to 80, and more specifically from 30 to 70, it is intended thatvalues such as 15 to 85, 22 to 68, 43 to 51, 30.3 to 32, etc., areexpressly enumerated in this specification. In other words, all possiblecombinations of numerical values between the lowest value and thehighest value enumerated are to be considered to be expressly stated inthis application in a similar manner.

EXAMPLES Example 1 In vitro Toxicology Studies

Cell viability was determined using human umbilical vein endothelialcells (HUVEC, first passage). Cells were treated with the active agentin doses ranging from 0.1 μM to 100 μM. The number of live and deadcells was determined using the MultiTox-Fluor assay (Promega) orLive/Dead® assay (Invitrogen). Logistic plots were used to determine thecompound's LD₅₀ value. Lipoic acid was not cytotoxic in theconcentration range.

Example 2 In vitro Efficacy Studies

Increase in Elasticity: Pairs of mouse lenses were incubated in medium200 supplemented with an antibiotic, an antimycotic, in the presence orabsence of lipoic acid (concentrations ranging from 0.5 μM to 500 μM)for 8-15 hours. Each lens was removed from medium, weighed, andphotographed on a micrometer scale. A coverslip of known weight(0.17899±0.00200 g) was placed on the lens, and the lens wasphotographed again on the micrometer scale. The diameter of each lenswith and without the coverslip was determined from the photographs. Thechange in lens diameter produced by the force (coverslip) was computedΔD=(D_(withcoverslip)−D_(withoutcoverslip)). The results (FIG. 4, ‡)indicate that lipoic acid at concentrations≥9.6 μM caused astatistically significant increase in ΔD, p<0.0001.

Decrease in disulfide bonds: Lipoic acid at concentrations≥9.6 μM causeda statistically significant decrease in protein disulfides in the mouselenses where there was a significant increase in ΔD (FIG. 4). Mouselenses were homogenized in a denaturing buffer containing a fluorescentalkylating agent to modify the free SH groups. After removing thealkylating agent homogenates were reduced and alkylated with a differentfluorescent alkylating agent. Absorption spectra of the modifiedproteins were used to calculate free protein SH and protein SS groups.The results are shown in FIG. 5.

Example 3 Syntheses of Lipoic Acid Choline Ester

Lipoic acid choline ester was prepared according to the followingsynthetic route. Choline salts of alternative reducing agents can besimilarly prepared by making the appropriate reagents substitutions.Also, one of ordinary skill in the art would recognize that thesesyntheses are provided as guidance and that reagents, conditions,amounts, temperatures, and the like may be modified without departingfrom the general synthetic pathway.

Step 1:

(R)-2-(dimethylamino)ethyl 5-(1,2-dithiolan-3-yl)pentanoate. A solutionof DCC (11 g. 53 mmol) in anhydrous CH₂Cl₂ (20 mL) was added withstirring over 10-20 minutes to a cold (0° C.) solution of R-lipoic acid(10.0 g, 48.5 mmol), N,N-dimethylethanolamine (14.5 mL, 145 mmol, 3eq.), and DM AP (600 mg, 4.9 mmol) in anhydrous CH₂Cl₂ (50 mL).Following complete addition, the cold bath was removed. After 18 hoursat room temperature, all volatiles were removed under reduced pressure,and the resulting residue was purified by flash column chromatography(SiO₂, 2% McOH in CH₂Cl₂) providing the desired product as a clearyellow oil (10.6 g, 79%). All data consistent with values reported inthe literature. (See Courvoisier C. et al. 2006. Synthesis and effectsof 3-methylthiopropanoyl thiolesters of lipoic acid, methanol metabolitemimics. Bioorganic Chemistry 34(1):49-58.)

Step 2:

(R)-2-(5-(1,2-dithiolan-3-yl)pentanoyloxy)-N,N,N-(trimethyl)ethylammouiumiodide, Methyl iodide (0.55 mL, 9.0 mmol) was added to a solution of theamine (2.5 g, 9.0 mmol) in anhydrous (CH₂Cl₂ (20 mL). The reactionmixture was stirred overnight and slowly poured into diethyl ether (250mL) with vigorous stirring. The choline salt was isolated by filtrationas a free-flowing pale, yellow sold (3.7 g, 98%).

Example 4 One-Step Synthetic Route

Example 5 Eye Drop Formulation of Lipoic Acid Choline Ester

The following eye drop formulation was prepared using lipoic acidcholine ester as the active agent.

Formula A

Concentration Ingredient % by weight Purpose Lipoic acid choline ester5.0 Active agent Ethyl pyruvate 0.1 Energy source Sodium phosphate 0.269Buffer monobasic monohydrate, USP Sodium phosphate 0.433 Buffer dibasicanhydrous, USP Sodium chloride 0.5 Tonicity agentHydroxypropylmethylcellulose 0.2 Viscosity agent (HPMC), USP De-ionized,pyrogen free water to 100 mL Solvent

Formula B

Concentration Ingredient % by weight Purpose Lipoic acid choline ester5.0 Active agent Alanine 0.5 Stabilizer Sodium phosphate 0.269 Buffermonobasic monohydrate, USP Sodium phosphate 0.433 Buffer dibasicanhydrous, USP Sodium chloride 0.5 Tonicity agentHydroxypropylmethylcellulose 0.2 Viscosity agent (HPMC), USP De-ionized,pyrogen free water to 100 mL Solvent

The eye drop formulation has a pH of 7.0.

The pharmaceutical formulation may be diluted to 100 ml filtered water(e.g., Millex syringe filter (0.45 micron 33 mm). The pharmaceuticalcomposition may be packaged for multi-dose administration, e.g., 2-7 mL(e.g., 5 mL) eyedropper bottle with screw lid dropper.

The examples given above are merely illustrative and are not meant to bean exhaustive list of all possible embodiments, applications, ormodifications of the invention. Thus, various modifications andvariations of the described methods and systems of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes ofcarrying out the invention which are obvious to those skilled in thechemical arts or in the relevant fields are intended to be within thescope of the appended claims.

The disclosures of all references and publications cited above areexpressly incorporated by reference in their entireties to the sameextent as if each were incorporated by reference individually.

What is claimed is: 1.-30. (canceled)
 31. A one-step method forpreparing a choline ester of a reducing agent, comprising reacting thereducing agent with a halogenated choline in a reaction mixture.
 32. Themethod according to claim 31, wherein the reducing agent is a lipoicacid or derivative of lipoic acid.
 33. The method according to claim 32,wherein the lipoic acid or derivative of lipoic acid is alpha lipoicacid, 6,8-dimercaptoctanoic acid, dihydrolipoate,5-(1,2-thiaselenolan-5-yl)pentanoic acid or5-(1,2-thiaselenolan-3-yl)pentanoic acid.
 34. The method according toclaim 31, wherein the reacting reaction is conducted in a solvent.
 35. .The method according to claim 34, wherein the solvent is acetone ordimethyl formamide (DMF).
 36. The method according claim 31, wherein thereaction mixture further comprises a base.
 37. The method according toclaim 36, wherein the base is selected from the group consisting ofK₂CO₃, Cs₂CO₃, KF, NaHCO₃, and KH₂PO₄.
 38. The method according to claim36, wherein the base is present in an amount of about 1 to about 5equivalents relative to the reducing agent.
 39. The method according toclaim 38, wherein the base is present in an amount of about 1 equivalentrelative to the reducing agent.